tbuckley/cabot-search huggingface dataset:
Abstract 4: A novel humanized mouse model for study of T cells and NK cells in the tumor microenvironment
Authors: Jyoti Arora, Caitlin D. Lemke-Miltner, George J. Weiner
Journal: Cancer Research (2023)

Abstract Humanized mouse models can be useful for in vivo preclinical evaluation of mechanisms underlying cancer immunotherapy. Most such models involve systemic engraftment of immunocompromised mice with human lymphoid and hematopoietic cells, often using normal donor peripheral blood mononuclear cells (PBMCs), followed by inoculation with human tumor. The ability to control and modify immune cells within the tumor microenvironment (TME), especially human NK cells, is limited in such models. Here, we report the development of a mouse model designed to control the number of human T cells and NK cells in the TME. 18 mice and pre-validated PBMCs were obtained from Charles River. Six groups of 3 mice each were inoculated with Raji lymphoma cells. Group 1 was inoculated with Raji cells only and received no PBMCs. Raji cells were mixed PBMCs for groups 2-4. Group 5 was systemically engrafted with PBMCs 12 days prior to Raji inoculation and Group 6 was systemically engrafted with PBMCs 10 days after Raji inoculation. All mice developed tumors with similar growth characteristics. Serial fine needle aspirates (FNAs) of the resulting tumors were evaluated by flow cytometry. Results of FNAs are summarized below. Tumors were harvested at the end of experiment, typically 27 days after tumor inoculation, and evaluated by immunohistochemistry. Both T cells and NK cells were evenly distributed within tumors at the time of harvest. We conclude co-injection of tumor and PBMCs results in engraftment of both T cells and NK cells within the TME for up to 27 days, with T cells and NK cells distributed evenly within the tumor. Mixing higher numbers of PBMCs with tumor cells resulted in increased numbers of T cells and NK cells in the TME throughout the experiment. Matrigel did not improve engraftment or persistence of T or NK cells in the TME. We conclude mixing PBMCs with Raji tumor cells prior to tumor inoculation into immunocompromised mice is an effective approach when a humanized model with human T cells and NK cells within the TME is desired. Group Description % T cells in tumor at 17 days % NK cells in tumor at 17 days 1 No PBMCs 0 0 2 PBMCs mixed with Raji at 10:1 ratio 8.6 1.5 3 PBMCs mixed with Raji at 2:1 ratio 20.3 2.7 4 PBMCs and Raji in matrigel 9.2 0.6 5 PBMCs systemically engrafted 12 days before Raji inoculation 23.2 0.1 6 PBMCs systemically engrafted 10 days after Raji inoculation 19.6 0.2 Citation Format: Jyoti Arora, Caitlin D. Lemke-Miltner, George J. Weiner. A novel humanized mouse model for study of T cells and NK cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4.
Abstract 672: A genetic defect in phenylalanine hydroxylase (PAH) affects the immune system in kidney
Authors: Seojeong Kim, Chaelin Kang, Jihyun Park, Seung Seok Han, Youngil Koh, Sung-Soo Yoon
Journal: Cancer Research (2023)

Abstract PAH, one of the genes associated with inborn error of metabolism (IEM), codes for phenylalanine hydroxylase and is specifically expressed in the liver and kidney. When PAH is dysfunctional, phenylalanine (Phe) cannot be converted to tyrosine (Tyr), and it produces toxic metabolites such as phenylpyruvate by a metabolic shift. We previously analyzed germline variants of IEM-related genes using public cancer multi-omics data. As a result, there was a high frequency of mutations in the PAH gene, and germline variants were particularly high in renal cell carcinoma (RCC). Based on this data, we suggest that kidney-specific expressed PAH mutations could change the kidney tissue microenvironment, which makes them more vulnerable to chronic kidney disease (CKD) and affects cancer development. We used the PAH enu2 mice model, which has a missense mutation in the PAH gene. PAH homozygous mutant mice display phenylketonuria (PKU) when not maintained on a Phe-free diet. PAH enu2 mice were maintained a normal diet. The concentration of phenylalanine in the mice's serum was measured each week. Then, the proportions of immune cells were analyzed in peripheral blood and kidney. Next, Jurkat cells, which are well-known as human T-cell lines, were applied to the in vitro experiment. Jurkat cells were cultured in a medium containing Phe or PPA and were activated by PHA/PMA or anti-CD3/CD28. Then after, the expression of immune-related markers was evaluated. Additionally, the expression of the PAH enzyme in human RCC and normal tissue was assessed by immunohistochemistry (IHC). The concentration of Phe in mice serum was higher when the PAH gene was defective; homozygous mutant, heterozygous mutant. As PAH-defective mice got older, the T cell population decreased in the blood and kidney immune cells. When Jurkat T cells were cultured with Phe or PPA, the expression of IL-2, a cytokine related to T cell expansion, decreased in the PPA-treated and activated group. There were more significant changes when treated with PPA rather than Phe. IHC data shows the expression of PAH was significantly decreased in RCC tissues compared to non-tumor tissue. These results suggest that the immune system could be changed by PAH dysfunction bearing a germline mutation. Moreover, the kidney tissue microenvironment with PAH defects is vulnerable to the development of kidney disease, which can lead to RCC. This is a novel discovery that can explain the importance of the role PAH gene in kidney disease. Citation Format: Seojeong Kim, Chaelin Kang, Jihyun Park, Seung Seok Han, Youngil Koh, Sung-Soo Yoon. A genetic defect in phenylalanine hydroxylase (PAH) affects the immune system in kidney [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 672.
Abstract 2341: Group 2 innate lymphoid cells influence pancreatic stromal cell composition in homeostasis and pancreatic cancer
Authors: Thomas Yip, Sheng Lee, Julie Stockis, Celine Garcia, Shwetha Raghunathan, Bram Lim, Silvain Pinaud, Shaun Png, Timotheus Halim
Journal: Cancer Research (2023)

Abstract Background: Group 2 Innate Lymphoid Cells (ILC2s) are tissue-resident innate immune cells critical in orchestrating type-2 immune responses. Cancer-associated fibroblasts are increasingly recognized as having important pro- and anti-tumorigenic roles. While type-2 immunity has been linked to fibrosis and tumor development, how ILC2s influence the stromal microenvironment in these settings is poorly understood. Here, we investigated the role of ILC2s on pancreatic stromal cell (PSC) behavior in healthy and tumor-bearing mice. Methods: Naive, inflamed, and tumor-bearing pancreata of WT or ILC2-deficient mice were profiled by flow cytometry, histology, or multiplex immunofluorescence imaging. We assessed two-way communication between ILC2s and stromal cells using in silico analysis of single-cell RNAseq or in vitro coculture transcriptomic data. Putative crosstalk mechanisms were tested with in vitro and in vivo assays. Additionally, we performed two-photon microscopy of reporter mice pancreata and multiplex immunofluorescent imaging of tumor sections. Results: Imaging studies show that ILC2s co-localize with IL-33+ stromal cells in the naïve, inflamed and tumor-bearing pancreas. In vivo and in vitro studies indicate that ILC2s are critical mediators of stromal cell proliferation and density in homeostatic and inflamed conditions, while influencing stromal cell-driven immune modulation in cancer. Single-cell transcriptomic analysis reveal distinct Il33+ and Il33- stromal cell clusters, which may engage in different crosstalk mechanisms with ILC2s, as determined by in silico inferential analysis, in vitro co-culture and in vivo studies. Conclusion: ILC2s strongly influence stromal cell composition in both the healthy pancreas and pancreatic tumors. Given the importance attributed to the characteristic desmoplasia of pancreatic tumors, ILC2-driven modulation of the stromal compartment may affect tumor progression. Citation Format: Thomas Yip, Sheng Lee, Julie Stockis, Celine Garcia, Shwetha Raghunathan, Bram Lim, Silvain Pinaud, Shaun Png, Timotheus Halim. Group 2 innate lymphoid cells influence pancreatic stromal cell composition in homeostasis and pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2341.
Abstract 419: Elevated Akt/mTOR signaling drives therapy resistance in ALM
Authors: Jeremy A. Bravo Narula, Vito W. Rebecca
Journal: Cancer Research (2023)

Abstract Metastatic acral lentiginous melanoma (ALM) is the most lethal subtype of cutaneous melanoma and effective therapies for patients have not been established. Studies revealed a link between the dysregulation of CDK4/6 and ALM mortality, however CDK4/6 inhibitors (CDK4i/6i) have proved ineffective in patients with advanced ALM. The tumor suppressor Rb was recently shown to have a novel suppressive role towards mTORC2. Here, we elucidate the role of a non-canonical tumor suppressor function of Rb in negative regulation of mTOR in CDK4i/6i-resistance. We hypothesize that the loss of Rb induced by CDK4i/6i triggers phosphorylation of Akt and downstream mTORC1 signaling pathways. We characterized baseline activity of the PI3K/AKT/mTOR pathway in a panel of ALM cell lines, finding heterogeneity across signaling. We immunoblotted for key proteins involved in the signal reprogramming hypothesized: pRb, pAkt, pS6 to define activation status and found the loss of Rb phosphorylation preceded the enrichment of pAKT, supporting the non-canonical role of Rb tumor suppression and the Rb-mTORC2 signaling axis as intrinsic CDK4i/6i mechanism. In parallel, we assessed the essential protein-protein interactions required for mechanistic mTORC1 activation—mTOR and Rheb GTPase. The enrichment of these interactions shortly after introducing CDK4i/6i supported our hypothesized mechanism. Translational utility of CDK4i/6i and mTORi dual inhibition was determined via long-term Colony Formation Assays (CFAs) using Palbociclib, rapamycin and sapanisertib (TAK228), a catalytic mTOR inhibitor. We generated Palbociclib (acquired)-resistant ALM cell lines and investigated the pharmacological inhibition of PI3K signaling pathways through immunoblot and colony-formation assays. Concurrent mTORi and CDK4i/6i inhibition showed effective reduction in pRb, pS6, Cyclin D and increases in Cleaved PARP protein, suggesting apoptotic cell death was elevated with combined CDK4i/6i+mTORi treatment. Similarly, CFAs revealed dual inhibition elicited superior antiproliferative effect over the monotherapy treatments. CFAs with acquired-resistance cell lines also showed that combination inhibition decreased colony formation, showing the potential utility of concurrent CDK4i/6i and mTORi as an approach against acquired resistance. Our results show the efficacy of combined inhibition as an anti-tumor strategy. Citation Format: Jeremy A. Bravo Narula, Vito W. Rebecca. Elevated Akt/mTOR signaling drives therapy resistance in ALM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 419.
Abstract 330: <i>EP300</i> loss drives tumorigenesis in bladder cancer via activation of IL-6/JAK/STAT3 signaling
Authors: Sizhi P. Gao, James A. Rodrigues, Amanda R. Sabel, Jiaqian Luo, Chen Ziyu, Xinran Tang, Eduardo A. Mascareno, Naryan Rustgi, Hikmat Al-Ahmadie, Kwanghee Kim, Eugene J. Pietzak, Gopakumar V. Iyer, David B. Solit
Journal: Cancer Research (2023)

Abstract Bladder cancer (BLCA) is the sixth most common cancer in the U.S with 83,730 new cases, accounting for 17,200 deaths in the year 2021 and 212,536 deaths worldwide. EP300, a histone acetyltransferase gene, is mutated in ~17% of BLCA and in other cancer types such as endometrial cancer. The mechanisms by which EP300 mutations contribute to tumorigenicity of BLCA is not currently understood. To determine the phenotypic effects of loss-of-function EP300 mutations, we generated isogenic BLCA cells with EP300 knocked out (KO) using CRISPR/CAS9. Conspicuous changes in phenotypes consistent with more aggressive forms of cancer were observed in EP300-null BLCA clones, including luminal-basal histotype plasticity, anchorage-independent growth, enhanced in vitro and in vivo cell proliferation and enhanced invasion in Boyden chamber assays. To identify mechanisms whereby EP300 KO promotes increased oncogenicity, we performed phospho Tandem Tag Mass Spectrometry (TMT MS) using lysates from parental and EP300 KO cells. A notable finding was significantly increased expression of phosphorylated STAT3 (pSTAT3), which we subsequently confirmed by western blot. siRNA knockdown and selective inhibitor experiments indicated JAK1 as the upstream activator of pSTAT3 in BLCA cells. Studies of conditioned media (CM) from the EP300 KO cells using ELISA and neutralizing antibodies demonstrated that secreted IL-6 ligand and soluble IL-6R in the CM drove STAT3 activation through the IL-6 family common signaling subunit, gp130, i.e. trans-signaling. BLCA RT112 cells express an oncogenic FGFR3-TACC3 fusion and are sensitive to the FDA-approved FGFR kinase inhibitor erdafitinib. pSTAT3 expression was not dependent on FGFR3 signaling in EP300 KO RT112 cells, and EP300 KO was sufficient to confer erdafitinib resistance in a BLCA context. In sum, our results uncover that EP300 loss enhances IL-6/JAK/STAT3 signaling which promotes BLCA tumorigenesis and FGFR inhibitor resistance. Our findings elucidate a role for cytokine induced sterile inflammation in EP300-mediated tumorigenesis in bladder cancers and suggest that targeting the IL-6/JAK/STAT3 axis may represent a novel therapy strategy to overcome FGFR3 inhibitor resistance. Citation Format: Sizhi P. Gao, James A. Rodrigues, Amanda R. Sabel, Jiaqian Luo, Chen Ziyu, Xinran Tang, Eduardo A. Mascareno, Naryan Rustgi, Hikmat Al-Ahmadie, Kwanghee Kim, Eugene J. Pietzak, Gopakumar V. Iyer, David B. Solit. EP300 loss drives tumorigenesis in bladder cancer via activation of IL-6/JAK/STAT3 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 330.
Abstract 2674: Synergistic combinations of lurbinectedin with irinotecan and ONC212 in pancreatic cancer
Authors: Tej Tummala, Arielle De La Cruz, Ash Uruchurtu, Nicholas R. Liguori, Abbas E. Abbas, Leiqing Zhang, Christopher G. Azzoli, Lanlan Zhou, Wafik S. El-Deiry
Journal: Cancer Research (2023)

Abstract Pancreatic cancer is a devastating disease with a five-year survival rate below 10% according to the American Cancer Society. Novel chemotherapeutics and targeted therapies in pancreatic cancer have shown limited success, illustrating the urgent need for new treatments. Lurbinectedin is a chemotherapeutic synthetic tetrahydroisoquinoline alkaloid that inhibits active transcription by binding to guanine rich sequences in the minor groove of DNA. Lurbinectedin has been shown to reduce oncogenic transcription by stalling and degrading RNA Polymerase II while also inducing single- and double-stranded breaks to DNA, causing subsequent apoptosis. Lurbinectedin received accelerated FDA approval in 2020 for metastatic small cell lung cancer on or after platinum-based chemotherapy and is currently undergoing clinical trials in a variety of tumor types. We recently described a synergistic interaction between lurbinectedin and ONC201/TIC10, a compound that induces the TRAIL pathway, in killing SCLC cell lines associated with activation of p-Chk1 and the integrated stress response. We now demonstrate lurbinectedin’s efficient killing of pancreatic tumor cells as a single agent in PANC-1, BxPC-3, and HPAF-II cell lines, with IC-50s corresponding to sub-nanomolar concentrations. We also demonstrate that a combination of lurbinectedin and irinotecan, a topoisomerase I inhibitor with FDA approval for advanced pancreatic cancer, results in synergistic killing of pancreatic tumor cells in vitro. We further demonstrate a combination of lurbinectedin and ONC212, an imipridone, the same class of compounds as ONC201, also results in synergistic killing of pancreatic tumor cells. Cell viability was measured using the CellTiterGlo assay at varying drug concentrations. We hypothesize a combination therapy of lurbinectedin and irinotecan or ONC212 can enhance immune cell killing of pancreatic tumor cells. This is being explored by co-culturing CD8+ T lymphoblast cells with pancreatic tumor cells treated with lurbinectedin, irinotecan, ONC212, and combination, along with molecular mechanisms of cell death and effects on cytokines in the tumor microenvironment. Our results are developing insights regarding molecular mechanisms underlying therapeutic efficacy of a novel combination drug treatment for pancreatic cancer. Citation Format: Tej Tummala, Arielle De La Cruz, Ash Uruchurtu, Nicholas R. Liguori, Abbas E. Abbas, Leiqing Zhang, Christopher G. Azzoli, Lanlan Zhou, Wafik S. El-Deiry. Synergistic combinations of lurbinectedin with irinotecan and ONC212 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2674.
Abstract 2582: Protein aggregation in sarcoma as a target for therapy
Authors: Sara Sartini, Hillary Le, Alice Soragni
Journal: Cancer Research (2023)

Abstract Sarcomas are rare and heterogenous cancers arising from bone, soft and connective tissue. Developing effective therapies remains a challenge, since many types are understudied and poorly characterized at the molecular level. Here, we propose to investigate the role of protein misfolding in sarcoma, starting from the tumor suppressor p53. Wild type p53 is a tumor suppressor that regulates many cellular processes and prevents malignant transformation. Missense mutations promote p53 misfolding and convert the protein into a cancer-promoting oncogene. Mutant p53 is found in ~80% of all osteosarcoma cases, ~50% of leiomyosarcoma and angiosarcomas. It is also mutated at lower frequencies in other sarcoma subtypes. We investigated whether p53 mutations found in different types of sarcoma cause p53 misfolding and aggregation and can be targeted by ReACp53, a p53 aggregation-targeting peptide with anti-tumor activity in ovarian cancer and prostate cancer (Soragni et al, 2016, Zhang et al, 2019). We established tumor organoids from four sarcoma lines, which include osteosarcoma, fibrosarcoma, leiomyosarcoma and rhabdomyosarcoma. The cells carry either p53 mutations or WT p53. To characterize the p53 aggregation status and elucidate the response to ReACp53, we performed staining and quantification of folded/unfolded p53 pools in the organoids. Our results demonstrate the presence of overexpression and aggregation of p53 in the nuclei as well in the cytosol of different types of sarcoma lines. We found a marked susceptibility to ReACp53 in all lines, including cells bearing WT p53. We also explored whether similar p53 conformational defects are found in tumor tissue from sarcoma patients and if the activity of ReACp53 is confirmed in patient-derived tumor organoids. Overall, the discovery of structural alterations of p53 in sarcoma could provide an opportunity for the development of novel therapeutic strategies. Citation Format: Sara Sartini, Hillary Le, Alice Soragni. Protein aggregation in sarcoma as a target for therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2582.
Abstract 5942: Mechanism and role of neutrophil recruitment in head and neck squamous cell carcinoma
Authors: Kulsoom Ghias, Hina Shakeel, Alisalman Sheikh, Syed Hani Abidi, Kiran I. Masood, Saira Fatima, Fareena Bilwani
Journal: Cancer Research (2023)

Abstract The immune system plays a pivotal role in identification and clearance of tumor cells, but can also be counterproductive and enhance tumor progression. Presence of neutrophils in solid tumors, including the sixth most common cancer globally - head and neck squamous cell carcinomas (HNSCC), is associated with poor prognosis. Pro-inflammatory cytokines regulated by NF-κB promote extravasation of neutrophils into tissue; neutrophils are polarized to anti-tumor (N1) or pro-tumor (N2) phenotype through the action of various pathways, including TGF-β. The mechanism and role of neutrophil recruitment in HNSCC were explored this study. Neutrophils isolated from adult donors were co-cultured for 5 hours with conditioned media (CM) from HNSCC cells SCC-9 and Cal-27, and stained with TNF-α, ICAM-1, CCL2, CCL5 for analysis of neutrophil phenotype by flow cytometry. TGF-β blocking antibody was used to explore the role of TGF-β in polarization of neutrophils. Canonical and non-canonical TGF-β signaling pathway activation was also investigated in these neutrophils through western blots. Using HNSCC patient samples, qPCR was performed for expression of genes involved in apoptosis, epithelial-to-mesenchymal transition, metastasis, and neutrophil function. Following co-culture with 24-hour HNSCC CM, neutrophils begin to exhibit cell surface markers indicative of N2 phenotype, which is impacted by TGF-β blocking antibody. Neutrophils co-cultured with HNSCC CM exhibited altered expression of TGF-β mediators Smad2/3, p38 and Akt, which was abrogated with the addition of TGF-β blocking antibody indicating specificity of a TGF-β induced signaling. Gene expression analysis exhibited significantly higher expression of anti-apoptotic BCL2 and neutrophil function marker alpha defensin 1 in HNSCC patient samples with greater neutrophil infiltration, while caspase 3 was expressed at significantly higher levels in HNSCC patient samples with low neutrophil infiltration. HNSCC cells recruit neutrophils; evidence suggests that TGF-β pathway, which typically promotes N2 phenotype in neutrophils, is involved via canonical and non-canonical mediators including p38 and Akt. Further, HNSCC patient samples have varying levels of neutrophil infiltration and differentially express genes involved in apoptosis, EMT, metastasis and invasion, inflammation. Citation Format: Kulsoom Ghias, Hina Shakeel, Alisalman Sheikh, Syed Hani Abidi, Kiran I. Masood, Saira Fatima, Fareena Bilwani. Mechanism and role of neutrophil recruitment in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5942.